Human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein gp160 is proteolytically cleaved into the surface subunit gp120 that is responsible for virus binding to the receptors and the transmembrane fusion protein subunit gp41 that mediates virus fusion and entry. The gp41 molecule contains a cytoplasm domain (CT), a transmembrane domain (TM), and an extracellular domain (ectodomain) which consists of three major functional regions: fusion peptide (FP), N-terminal heptad repeat (NHR), and C-terminal heptad repeat (CHR). Both NHR and CHR regions are composed of 4-3 hydrophobic heptad repeat (HR) sequences which have a tendency to form coiled coil structure.
During HIV infection, gp120 binds to CD4 and a chemokine receptor (CCR5 or CXCR4) on the target cell to trigger gp41 structural rearrangement. This results in the formation of a stable gp41 six-helix bundle (6-HB) core structure, in which three NHR-helices associate to form the central trimeric coiled coil. Three C-helices pack obliquely in an anti-parallel manner into the highly conserved hydrophobic grooves on the surface of the NHR-trimer. In each groove, there is a highly conserved hydrophobic deep pocket formed by the pocket-forming sequence (residues 565-581) in the NHR region. This pocket plays a critical role in viral fusion and maintaining the stability of the six-helix bundle. The formation of the six-helix bundle is believed to bring both the viral and target cell membranes into proximity, resulting in fusion between the virus and target cell membranes.
In the early 1990s, the first highly potent anti-HIV peptide, SJ-2176 (SEQ ID NO:3) was identified from the HIV-1 gp41 CHR region. Later, an analogous anti-HIV-1 peptide, T-20 (SEQ ID NO:29), was reported. In 2003, the US FDA approved the T-20 peptide (generic name: enfuvirtide; brand name Fuzeon®) as the first member of a new class of anti-HIV drugs—HIV fusion/entry inhibitors, which block HIV fusion with and entry into the target cell.
T-20 is effective as a salvage therapy for HIV/AIDS patients who have failed to respond to current antiretroviral therapeutics, including reverse transcriptase inhibitors (RTIs) and protease inhibitors (PIs). However, T-20 has several weaknesses. Firstly, it lacks oral bioavailability, resulting in an inconvenient dosage form and schedule, a significant barrier to patient acceptance and adherence. Secondly, its potency is not high. Thirdly, this peptidic drug can be easily degraded by proteolytic enzymes in the blood, leading to its short half-life in vivo (about 2 hours). Because of these problems, T-20 must be maintained in the blood of HIV/AIDS patients at a constant high concentration. Therefore, T-20 has to be administrated by injection twice a day at 90 mg/dose, resulting in painful injection-site reactions in most patients and high cost to the patients (>$20,000/year/patient). Furthermore, T-20 can easily induce drug resistance, resulting in increasing failure rates in T-20-treated patients. To overcome the above limitations of T-20, several approaches have been conducted, including modification of T-20 sequence or structure, designing of recombinant proteins interacting with the gp41 NHR or CHR regions, and identification of small molecule HIV-1 entry inhibitors targeting gp41.
HIV has an inherent tendency to mutate and may become resistant to any anti-HIV drugs. Patients with drug-resistant strains have an increasing risk of treatment failure with subsequent treatment regimen. Therefore, it is essential to develop new drugs with mechanisms of action or resistance profiles different from the current anti-HIV drugs. Application of existing drugs in combination therapies could improvevirologic response and reduce probability for viral mutations, or slow the development of drug resistance.
Clinical applications of antiretroviral drugs with different targets in combinations have shown significant synergism in inhibiting HIV-1 infection, reducing adverse effects and delaying the emergence of drug resistance. Therefore, a combination of two or more HIV fusion/entry inhibitors with different targets or different mechanisms of action may have the following advantages: 1) to maximize anti-HIV activity and to sustain effective anti-HIV concentrations for longer time because of synergistic effects; 2) to minimize potential toxic effects, reduce the amount and frequency of drug use, and decrease the cost to patient, due to dose reduction; and 3) to have complementary or cooperative anti-HIV activity.